The inventors found that, simply by transferring a plasmid (hereinafter called “IR/MAR plasmid”) including a mammalian replication initiation region (IR; Initiation Region) and a mammalian nuclear matrix attachment region (MAR; Matrix Attachment Region) to a human-derived cancer cell (COLO 320 colon cancer cell strain and HeLa cell strain) by a lipofection method and then selecting the cell by utilizing a drug resistance gene (Blasticidin or Neomycin) being present on the plasmid, it is possible to:
(1) increase a copy number of a gene inside the cell up to approximately 10,000 copies, the gene encoding a protein to be expressed (hereinafter, the gene is referred to as “target gene”, as needed), and
(2) highly amplify the target gene in either cases where the target gene and the IR/MAR plasmid are transferred in the same gene construct (cis) or where the target gene and the IR/MAR plasmid are transferred respectively in different gene constructs (trans) (refer to Patent Literature 1 and Non Patent Literature 1). Based on the findings, the inventors realized a system (hereinafter called “high gene amplification system”), which makes it possible to amplify the target gene up to approximately 10,000 copies, simply by performing the steps of: transferring the IR/MAR plasmid and the target gene to the mammalian cell (e.g., human-derived cancer cell (COLO 320 colon cancer cell strain and HeLa cell strain) and CHO cell) by the lipofection method; and selecting the cell by utilizing the drug resistance gene (Blasticidin or Neomycin) being present on the plasmid.
FIG. 1 illustrates a mechanism on how a DM and HSR are generated by the IR/MAR plasmid (also called “IR/MAR vector”). The IR/MAR plasmids are bound as direct repeats within a host cell, so as to form a multimeric complex thereof (Step 1). The multimeric complex is stably present in the host cell and is replicated autonomously while the cell is growing. The multimeric complex is cytogenetically recognized as a DM when the multimeric complex grows to a large size, or when the multimeric complex is integrated into a preexisting DM within the host cell. Further, as illustrated in Step 2, a double strand of a circular DNA of the multimeric complex is broken (DSB; double strand breakage) within the host cell, thereby the circular DNA turns into a linear DNA. Then, the linear DNA is integrated into a chromosome. Then a BFB (Breakage-Fusion-Bridge) cycle as illustrated in Step 3 is initiated, thereby triggering HSR generation.